Nano-encapsulation using GRAS materials and applications thereof

ABSTRACT

In one aspect, methods of preparing composite nanoparticle compositions are described herein. For example, in some embodiments, a method comprises providing a zein solution stream, an organic fluid stream including at least one additive and at least one buffer fluid stream. The zein solution stream, organic fluid stream and buffer fluid stream are delivered to a chamber for mixing at one or more rates sufficient to flash precipitate composite nanoparticles including the additive encapsulated by a shell comprising the zein.

RELATED APPLICATION DATA

This application is a U.S. National Phase of PCT/US2016/046052, filedAug. 8, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 62/203,167 filed Aug. 10, 2015,each of which are incorporated herein by reference in their entireties.

FIELD

The present invention relates to compositions fabricated by flashnanoprecipitation (FNP) and, in particular, to nanoparticle compositionsemploying generally recognized as safe (GRAS) materials.

BACKGROUND

Modulating individual monomer structures in synthetic polymers enablesthe design of nanoparticles with specific encapsulation and releasecharacteristics, but many synthetic polymers are still subject to FDAapproval and their cost can add substantially to the price of therapy.Biodegradable protein polymers, such as albumin, casein, gelatin, andchitosan, among others, have been investigated as all-natural low-costalternatives.

Zein is a prolamin protein found in endoplasmic reticulum-derivedprotein vesicles of maize seeds and generally finds application as filmcoating excipient of pharmaceuticals. Zein is water-insoluble owing toits high content (>50%) of non-polar amino acids such as leucine,proline, alanine, and phenylalanine. Globular zein consists of fourfractions that vary in molecular weight, composition, structure andsolubility. These include α-zein (MW, 19-24 kDa; 75-80% of totalprotein), β-zein (17-18 kDa, 10-15%), γ-zein (27 kDa, 5-10%), and δ-zein(10 kDa).

The zein subcomponents are arranged into a tertiary structure thatcomprises nine homologous repeating units oriented in an anti-parallelsense and stabilized by hydrogen bonds. The majority of the molecularsurface area comprises the hydrophobic α-helixes in anti-parallelorientation, while the glutamine rich turns create a hydrophilic surfaceat their top and bottom. Together, this assembly bestows zein withamphiphilic characteristics. These properties are reported to driveself-assembly into a variety of mesostructres including ribbons, sheets,tori, pores, and micro- and nanospheres. These structures were achievedmainly by either solvent evaporation or anti-solvent precipitation. Zeincolloidal particles are water-insoluble and overall hydrophobic, whichhas limited their application across industries. Driven by hydrophobicassociation they may aggregate or form precipitates.

A variety of techniques have been employed to produce particulate zeinstructures. Nanoparticles, for example, have been formed byemulsion-stripping processes, which are inherently expensive, difficultto scale from laboratory to industrial scale, and result in somewhatbroad particle size distributions because the initial emulsificationproduces a broad drop size distribution. Spray drying has been used, butleads to particles outside the desired nanoparticle size range, andleads to aggregation. While supercritical processing has beendemonstrated, larger sized particles are produced, and the cost ofsupercritical processing precludes its application in low-costcommercial production.

In view of these disadvantages, new fabrication processes are requiredenabling control and reproducibility of zein nanoparticle architectureson the industrial scale.

SUMMARY

In one aspect, methods of preparing composite nanoparticle compositionsare described herein. For example, in some embodiments, a methodcomprises providing a zein solution stream, an organic fluid streamincluding at least one additive and at least one buffer fluid stream.The zein solution stream, organic fluid stream and buffer fluid streamare delivered to a chamber for mixing at one or more rates sufficient toflash precipitate composite nanoparticles including the additiveincorporated into the composite nanoparticle structure. In someembodiments, the additive is encapsulated by or incorporated in a shellcomprising the zein. The additive, in some embodiments, is hydrophobic.Moreover, the zein solution stream, organic fluid stream and bufferfluid stream can be delivered to the chamber by independent feed lines,in some embodiments. Further, the zein fluid stream and/or buffer fluidstream can also comprise one or more stabilizers. In such embodiments,the stabilizers can be incorporated into the composite nanoparticlestructure. Generally, the composite nanoparticles can exhibit an averagesize of 10 nm to 500 nm with polydispersity less than 0.15 or less than0.1.

Alternatively, a method of zein nanoparticle fabrication comprisesproviding a zein solution stream and at least one organic fluid stream,wherein the zein solution stream and organic fluid stream are deliveredto a chamber for mixing at one or more rates sufficient to flashprecipitate zein nanoparticles into the organic fluid stream.Importantly, the zein nanoparticles exhibit a hydrophilic interior andhydrophobic exterior. This is in contrast to the preceding methodwherein hydrophobic moieties of the zein are oriented to thenanoparticle interior for interaction with the additive(s) encapsulatedby the zein. Zein nanoparticles having a hydrophilic interior can alsodisplay an average size of 10 nm to 500 nm with polydispersity of lessthan 0.3.

In additional embodiments, a method of zein nanoparticle fabricationcomprises providing a zein solution stream, at least one buffer fluidstream and at least one aqueous fluid stream. One or more additives areincluded in the buffer and/or aqueous fluid streams. The one or moreadditives can be hydrophilic in some embodiments. The zein solutionstream, aqueous stream and buffer fluid stream are delivered to achamber for mixing at one or more rates sufficient to flash precipitatecomposite nanoparticles including the additive incorporated into thecomposite nanoparticle structure. In some embodiments, the one or moreadditives are encapsulated by or incorporated in a shell comprising thezein. The zein fluid stream, aqueous stream and buffer fluid stream canbe delivered to the chamber by independent feed lines. Further, the zeinfluid stream and/or buffer fluid stream can also comprise one or morestabilizers. In some embodiments, the stabilizers can be incorporatedinto the composite nanoparticle structure. Generally, the compositenanoparticles formed according to this method can exhibit an averagesize of 10 nm to 500 nm with polydispersity less than 0.15 or less than0.1.

In a further aspect, methods of treating bacterial infections aredescribed herein. A method of treating a bacterial infection comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a composition comprising nanoparticles having a core-shellarchitecture, the core including one or more anti-bacterial agents andthe shell comprising zein. The core-shell nanoparticles, in someembodiments, can be prepared according to methods described hereinwherein the anti-bacterial agent is the additive encapsulated by a shellcomprising zein.

These and other embodiments are described in further detail in thedetailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) is a schematic of a method and associated apparatus accordingto some embodiments described herein.

FIG. 1(b) is a transmission electron microscopy (TEM) image of compositezein nanoparticles according to some embodiments described herein.

FIG. 2 illustrates nanoparticle size control according to someembodiments of methods described herein.

FIG. 3(a) illustrates size and PDI of zein nanoparticles without a lipidco-core relative to surfactant identity according to some embodimentsdescribed herein.

FIG. 3(b) illustrates size and PDI of composite nanoparticles with alipid co-core relative to surfactant identity according to someembodiments described herein.

FIG. 3(c) illustrates the effect of VitE-Ac incorporation on compositenanoparticle size as a function of mass relative to protein contentaccording to some embodiments described herein.

FIG. 3(d) illustrate the effect of sodium caseinate mass on compositenanoparticle size according to some embodiments described herein.

FIG. 4 illustrates composite nanoparticle size and PDI stability atvarious temperatures according to some embodiments described herein

FIG. 5 illustrate molecular structures and characteristics ofencapsulated dyes and autoinducer drug according to some embodimentsdescribed herein.

FIG. 6(a) illustrates composite nanoparticle size relative to the dyeidentity encapsulated according to some embodiments described herein.

FIG. 6(b) illustrates mean composite nanoparticle diameter relative todye encapsulation by weight according to some embodiments describedherein.

FIGS. 6(c) and 6(d) illustrate fluorescence excitation and emissionspectra respectively of composite nanoparticles encapsulating dyeaccording to some embodiments described herein.

FIG. 7 illustrates Nile Red encapsulation effects and encapsulationefficiency according to some embodiments described herein.

FIG. 8 illustrates zein nanoparticle size distribution according to someembodiments described herein, the zein nanoparticles having ahydrophilic interior and hydrophobic exterior.

FIG. 9(a) illustrates effect of composite nanoparticle size and PDI oftwo-fold and three-fold protein and VitE-Ac masses at constant CAI-1content according to some embodiments described herein.

FIG. 9(b) illustrates individual contribution of composite nanoparticleformulation components and their effect on size and PDI of resultingcolloids according to some embodiments described herein.

FIG. 10(a) illustrates bioluminescence response of V. cholerae WN1102supplemented with varying amounts of CAI-1-loaded Zein/CAS/VitE-Accomposite nanoparticles in PBS according to some embodiments describedherein.

FIG. 10(b) illustrates growth of V. cholera cultures at 37° C. inresponse to addition of CAI-1-loaded Zein/CAS/VitE-Ac nanoparticlesaccording to some embodiments described herein.

FIG. 11 illustrates DNA encapsulation and loading efficiency into zeincontaining colloids according to some embodiments described herein.

FIG. 12 illustrates BSA-FTIC encapsulation and loading efficiency intozein containing colloids according to some embodiments described herein.

FIG. 13 illustrates the effect of additives or stabilizers, having anisoelectric point below solution pH, on the size and PDI of resultingcolloids according to some embodiments described herein.

FIG. 14 illustrates the effect of additives or stabilizers, having anisoelectric point below solution pH, on the zeta potential of theresultant nanocarrier colloids according to some embodiments describedherein.

FIG. 15 characterizes solution stability of composite nanoparticles ofvarious construction.

FIG. 16 characterizes solution stability of composite nanoparticles ofvarious construction.

FIG. 17 characterizes solution stability of zein/casein nanoparticles.

FIG. 18 characterizes several properties of composite nanoparticles ofvarious construction and composition.

FIG. 19 provides compositions and flow rates for forming nanocarrierspecies of FIG. 9(a) according to methods described herein.

DETAILED DESCRIPTION

Embodiments described herein can be understood more readily by referenceto the following detailed description and examples and their previousand following descriptions. Elements, apparatus and methods describedherein, however, are not limited to the specific embodiments presentedin the detailed description and examples. It should be recognized thatthese embodiments are merely illustrative of the principles of thepresent invention. Numerous modifications and adaptations will bereadily apparent to those of skill in the art without departing from thespirit and scope of the invention.

I. Composite Nanoparticles

In one aspect, methods of preparing composite nanoparticle compositionsare described herein. As the composite nanoparticle compositions canfunction as carriers for various additive species, the nanoparticlecompositions can be considered nanocarriers. In some embodiments, amethod comprises providing a zein solution stream, an organic fluidstream including at least one additive and at least one buffer fluidstream. The zein solution stream, organic fluid stream and buffer fluidstream are delivered to a chamber for mixing at one or more ratessufficient to flash precipitate composite nanoparticles including theadditive incorporated into the composite nanoparticle structure. In someembodiments, the additive is encapsulated by a shell comprising thezein.

Turning now to specific steps, a zein solution stream is provided. Asdescribed in the examples herein, zein can be sourced from commercialsources. Alternatively, zein can be extracted from corn by a variety ofmethods such as those described in Shulka et al., Zein: the industrialprotein from corn, Industrial Crops and Products 13 (2001) 171-192.Moreover, zein employed in the solution stream can also include zeinderivatives. For example, zein can be modified to alter solubilityand/or other properties without affecting formation of compositenanoparticles according to methods described herein. Any solvent notinconsistent with the objectives of the present invention can be usedfor the zein solution stream. In some embodiments, a hydroalcoholicsolution serves as suitable solvent. Further, zein can be present in thesolution in any amount not inconsistent with the objectives of thepresent invention. For example, zein can be present in an amountselected from Table I.

TABLE I Zein amount (mg/mL)  1-10 2-9 4-8  5-10 ≥10

In addition to the zein solution stream, an organic fluid stream isprovided comprising at least one additive. Any organic fluid compatiblewith methods described herein can be employed. Organic fluid may varydepending on chemical identity of the one or more additives. In keepingwith the GRAS characteristics of the present methods, suitable organicfluid can be ethanol. In other embodiments, hydrophobic solvents such asTHF, DMSO, DMF or acetone may be employed. Further, a number ofadditives are contemplated, including hydrophobic additives andhydrophilic additives. Additive(s), for example, can be selected frompharmaceutical compositions, nutraceutical compositions, agriculturalcompositions, food and/or beverage compositions, taste-makingcompositions, biomolecular compositions and/or cosmetic compositions. Insome embodiments, compositions include antibacterial agents,anti-parasitic agents, immunosuppressive agents, immunoactive agents,anticoagulants, antiviral agents, agro actives such as pesticides,fungicides, insecticides and fertilizers, diagnostic agents, imagingagents, dyes, anti-cancer agents, anti-oxidants, preservatives,vitamins, neutraceuticals and combinations thereof. Biomolecularcompositions can include peptides, proteins, nucleic acids, nucleic acidfragments and combinations thereof. In some embodiments, nanoparticlecompositions can be employed for taste-masking of undesirable taste ofsubstances, such as bitter compounds. Non-limiting examples of agroadditives, nutraceuticals and taste-masking agents contemplated forencapsulation can include:

Taste- Agro Additive Nutraceutical masking Agent Isofetamdid Polyphenols(e.g. catechin, Quinine Azoxystrobin kaempferol, quercetin, etc.)Absinthin Boscalid Vitamins (e.g. Vit E, D, Papaverine Triademafon K andderivatives s.a. Cinnamedrine Triticonazole alpha tocopherol etc.)Orphenadrin Tebucanazole Phospholipids Haloperidol PyraclostrobinCarotenoid Yohimbine Propriconazole Fatty acid (e.g. Omega 3, AdhumuloneTrifloxystrobin a-linolenic acid (ALA), Diphenidol Penthiopyrad EPA, DHAetc.) Methadone Carbosulfan Phytostanol (e.g. Isoxanthohumol Etofenproxcampesterol, ergosterol, Falcarindiol Ethiprole sitosterol, etc.)Xanthohumol Carbofuran Curminoids Consign Imidacloprid (e.g. Curcumin,etc.) Dicyclomine 4-(2,4-dichlorophenoxy) Ubiquinone, etc. Colupulonebutyrate DPBA 2-(3- chlorophenoxy)propionate CPPA PendimethalinPheromones, s.a Methyl eugenol

As described herein, at least one buffer stream is also provided. Anysuitable buffer composition not inconsistent with the objectives of thepresent invention can be employed. In some embodiments, pH of the bufferstream ranges from 7.0 to 8.0.

The zein solution stream, organic fluid stream and buffer stream aredelivered to a chamber for mixing at one or more rates sufficient toflash precipitate composite nanoparticles comprising the additive(s)encapsulated by a shell comprising the zein. In some embodiments, theadditive(s) are completely encapsulated by the shell comprising zein. Inother embodiments, the additive(s) are partially encapsulated by theshell comprising zein. The zein solution stream, organic fluid streamand buffer fluid stream can be delivered to the mixing chamber atsubstantially the same rate. Alternatively, the solution streams can bedelivered to the mixing chamber at differing rates. In some embodiments,for example, the solution streams are delivered to the mixing chamberaccording to Table II.

TABLE II Stream Delivery Rates Fluid Stream Delivery Rate (mL/min) ZeinSolution 10-20 Organic 10-20 Buffer 30-40The mixing apparatus can employ independent feed lines and pumps fordelivery of the individual fluid streams to the chamber. In someembodiments, the zein fluid stream, organic fluid stream and bufferfluid stream are simultaneously mixed in the chamber for flashprecipitation of the nanoparticles. Alternatively, the fluid streams canenter the mixing chamber in any desired order resulting in compositenanoparticle production. Advantageously, methods described herein canpermit continuous production of composite nanoparticles or batchproduction of composite nanoparticles. Suitable multi-inlet vortexmixing (MIVM) apparatus for methods described herein are described inU.S. Pat. No. 8,137,699 which is incorporated herein by reference in itsentirety. In some embodiments, fluid stream enter the mixing chamber atvelocities described in U.S. Pat. No. 8,137,699.

In some embodiments, the zein solution stream and the buffer fluidstream further comprise one or more stabilizers. Suitable stabilizerscan include one or more surfactants, such as alkyl-oxide copolymers,polygycols and/or proteins, poloxamers and phospholipids, non-ionicsurfactants, ionic surfactants, ionic- and nonionic lipids. Othersurfactants that may be used in the method include other nonionicsurfactants such as poloxamers (Pluronic), polyoxyethylene alkyl ethers(Brij), sorbitan esters (Span), polyoxyethylene sorbitan fatty acidesters (Tween), and ionic surfactants such as sodium dioctylsulfosuccinate, sodium lauryl sulfate, benzalkonium chloride, cetyltrimethyl ammonium bromide, n-dodecyl trimethyl ammonium bromide, andpolymer such as polyvinyl alcohol, polyvinyl pyrrolidone. Phospholipidsthat may be used in the method include non-ionic and charged lipids orphospholipids such as egg lecithin, soy lecithin, phosphatidyl choline,phosphatidyl ethanolamine, 1,2-dioleoyl-3-trimethyl ammonium propane. Byway of example only, the surfactant or stabilizer may be Polysorbate 80,Polysorbate 60, copolymer condensates of ethylene oxide and propyleneoxide, alpha-Hydro-omega-hydroxypoly (oxyethylene)poly(oxypropylene),poly(oxyethylene) block copolymers, methyl glucoside, coconut oil ester,poloxalene, lecithin, hydroxypropylmethyl cellulose, casein, sodiumcaseinate, calcium caseinate, chitosan hydrochloride (CHC), tocopherolpolyethylene glycol 1000 succinate, carboxymethyl cellulose, sorbitol,and glycerol. Examples of surfactants derived from natural plant oilsare ethoxylated coca oils, coconut oils, soybean oils, castor oils, cornoils and palm oils. A surfactant and/or stabilizer can be or can bederived from a plant extract or a biodegradable plant extract. Many ofthese natural plant oils are U.S. FDA GRAS (Generally Recognized AsSafe).

In some embodiments, for example, surfactant of the zein solution streamand/or buffer fluid stream is selected from tocopheryl polyethyleneglycol succinate (TPGS), casein and ethylene oxide/propylene oxidesurfactants under the PLURONIC® trade designation from BASF of FlorhamPark, N.J. Surfactant of the zein solution stream and/or buffer fluidstream can be incorporated into the composite nanoparticle structure.For example, surfactant can be incorporated into the hydrophobic core ofthe nanoparticle. Alternatively, surfactant can be incorporated intoand/or onto the shell comprising zein. In further embodiments,surfactant can be incorporated into the core, shell and onto shellsurfaces of the composite nanoparticles.

FIG. 1(a) is a schematic of a method and associated MIVM apparatusaccording to some embodiments described herein. As illustrated in FIG.1(a), the zein solution stream, organic fluid stream and buffer fluidstreams are delivered by independent lines to a vortex mixing chamberfor the continuous production of composite nanoparticles. The zeinsolution stream and buffer stream contain surfactant that isincorporated into the core, shell and/or onto shell surfaces of thecomposite nanoparticles.

Alternatively, a method of zein nanoparticle fabrication comprisesproviding a zein solution stream, at least one buffer fluid stream andat least one aqueous fluid stream. One or more additives are included inthe buffer and/or aqueous fluid streams. In some embodiments, forexample, the additive(s) are hydrophilic including, but not limited to,hydrophilic additives listed in this Section I above. As set forth inthe non-limiting examples herein, hydrophilic additive can includeproteins, nucleic acids, nucleic acid fragments and/or combinationthereof.

The zein solution stream, aqueous stream and buffer fluid stream aredelivered to a chamber for mixing at one or more rates sufficient toflash precipitate composite nanoparticles including the additive(s)incorporated into the composite nanoparticle structure. In someembodiments, the one or more additives are fully or partiallyencapsulated by a shell comprising the zein. The zein fluid stream,aqueous stream and buffer fluid stream can be delivered to the chamberby independent feed lines. Further, the zein fluid stream and/or bufferfluid stream can also comprise one or more stabilizers. In someembodiments, the stabilizers can be incorporated into the compositenanoparticle structure. In some embodiments, the solution streams aredelivered to the mixing chamber according to Table III.

TABLE III Stream Delivery Rates Fluid Stream Delivery Rate (mL/min) ZeinSolution 10-20 Aqueous 30-40 Buffer 10-40

Composite nanoparticles produced according to methods described hereinhaving a hydrophobic or hydrophilic additive encapsulated by a shellcomprising zein generally exhibit an average size of 10 nm to 500 nm. Insome embodiments, the composite nanoparticles have an average size of 40nm to 400 nm. Advantageously, the nanoparticles can have apolydispersity (PDI) of less than 0.15 or less than 0.1 in the as-formedstate. Moreover, the nanoparticles can maintain a polydispersity of lessthan 0.15 or less than 0.1 in a variety of liquid carriers, includingaqueous or aqueous-based liquid carriers. Fine control of nanoparticlesize and polydispersity enables methods and associated nanoparticlesdescribed herein to find application in protein size standards.Additionally, the composite nanoparticles can comprise up to 60 wt. % ofadditive(s). In some embodiments, a composite nanoparticle has anadditive loading selected from Table IV.

TABLE IV Composite Nanoparticle Additive Loading (wt. %) 0.05-60  0.1-30   1-20 0.1-10  0.2-5   0.5-5  Moreover, methods described herein can generally exhibit additiveencapsulation efficiency of 40-100 percent or 40-95 percent. In someembodiments, methods can exhibit additive encapsulation efficiencygreater than 90%. In some embodiments, additive encapsulation efficiencyis selected from Table V.

TABLE V Additive Encapsulation Efficiency (%) ≥95 ≥97 ≥98 90-100II. Zein Nanoparticles

In another aspect, methods of fabricating zein nanoparticles aredescribed herein. For example, a method of zein nanoparticle fabricationcomprises providing a zein solution stream and at least one organicfluid stream, wherein the zein solution stream and organic fluid streamare delivered to a chamber for mixing at one or more rates sufficient toflash precipitate zein nanoparticles into the organic fluid stream.Importantly, the zein nanoparticles exhibit a hydrophilic interior andhydrophobic exterior. This is in contrast to the preceding method ofSection I wherein hydrophobic moieties of the zein are oriented to thenanoparticle interior for interaction with the hydrophobic additive(s)encapsulated by the zein. Zein nanoparticles having a hydrophilicinterior can also display an average size of 10 nm to 500 nm withpolydispersity of less than 0.3.

Turning now to specific steps, a zein solution stream is provided. Thezein solution stream can be prepared in accordance with the disclosurein Section I above. Zein and/or zein derivatives, in some embodiments,are solubilized in a hydroalcoholic solution. Further, the amount ofzein in the solution stream can be selected from Table I above. In someembodiments, zein may be modified to further enhance the hydrophiliccharacter of the nanoparticle interior.

In addition to the zein solution stream, an organic fluid stream isprovided. In keeping with the GRAS characteristics of the presentmethods, suitable organic fluid can be ethanol. In some embodiments,several organic fluid streams are provided. The zein solution stream andorganic fluid stream(s) are delivered to a chamber for mixing at one ormore rates sufficient to flash precipitate zein nanoparticles into theorganic fluid stream. In some embodiments, for example, the solutionstreams are delivered to the mixing chamber according to Table VI.

TABLE VI Stream Delivery Rates Fluid Stream Delivery Rate (mL/min) ZeinSolution 10-20 Organic 10-50As in Section I, the mixing apparatus can employ independent feed linesand pumps for delivery of the individual fluid streams to the chamber.In some embodiments, the zein fluid stream and organic fluid stream aresimultaneously mixed in the chamber for flash precipitation of thenanoparticles. Alternatively, the fluid streams can enter the mixingchamber in any desired order resulting in composite nanoparticleproduction. Advantageously, methods described herein can permitcontinuous production of composite nanoparticles or batch production ofcomposite nanoparticles. Suitable MIVM apparatus are described in U.S.Pat. No. 8,137,699. In some embodiments, the apparatus illustrated inFIG. 1(a) can be employed wherein three of the feed lines are ethanoland the remaining feed line the zein solution.III. Methods of Treating Infection

In a further aspect, methods of treating bacterial infections aredescribed herein. A method of treating a bacterial infection comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a composition comprising nanoparticles having a core-shellarchitecture, the core including one or more anti-bacterial agents andthe shell comprising zein. The core-shell nanoparticles, in someembodiments, can be prepared according to methods described in Section Iwherein the anti-bacterial agent is the hydrophobic additiveencapsulated by a shell comprising zein. As detailed in the examplesbelow, the anti-bacterial agent can be suitable for treating intestinalinfections, such as cholera. Composite nanoparticle described herein, insome embodiments, demonstrate instability in bile salts, therebyenhancing drug delivery to environments containing such salts.

These and other embodiments are further illustrated by the followingnon-limiting examples.

Example 1—Fabrication and Characterization of Composite NanoparticleCompositions

Methods

Materials

Zein from maize (Z3625), casein sodium salt from bovine milk (C8654),Pluronic F-68 (P7061), α-Tocopheryl Acetate (VitE-AC) (T3001), Nile Red,(72485), Pyrene 98% (185515), and Citric acid (77929) were purchasedfrom Sigma Aldirch (St. Louis, Mo.). Fisher BioReagents LB broth(BP9723) was used at 1× concentration. Sodium Citrate (F 0000-00-3) waspurchased from Mallinckrodt. α-tocopheryl poly ethylene glycol 1000succinate (TPGS 1000) was purchased from Eastman, and methyl red(M29610) from Fisher. CAI-1 was provided by the Semmelhack ResearchGroup, Department of Chemistry, Princeton University. Reagents were ofanalytical grade. Ethanol (Fisher, BP 2818-4) was used as co-solvent forzein. NP preparation used milli-Q water, purified by reverse osmosis,ion-exchange and filtration.

Preparation of Surfactant Stabilized Zein Nanoparticles

Zein was suspended at 6 mg/mL in 60% EtOH. Surfactants TPGS or PluronicF68, were added at 2% wt. relative to zein and the suspension sonnicatedfor 10 min. The zein-surfactant solution was rapidly mixed (12 mL/min)against an α-tocopheryl acetate (1 mg/mL in 100% EtOH, 12 mL/min) streamand a citrate buffer stream pH 7.5 (10 mM sodium citrate, 1.6 μM citricacid, 36 mL/min) using the MIVM mixer (FIG. 1), previously described.Flow rates were controlled by two Harvard Apparatus PHD2000 syringepumps. The resulting solution contained 20% EtOH (v/v) and a zein/VitEratio of 6:1 by mass.

Preparation of Casein Stabilized Zein Nanoparticles

Casein was dissolved at 1 mg/mL in citrate buffer pH 7.5 (10 mM sodiumcitrate, 1.6 μM citric acid) and sonnicated for 10 min. Zein wasdissolved at 6 mg/mL in 60% EtOH and sonnicated. Casein (36 mL/min) andzein (12 mL/min) were rapidly mixed against an α-tocopheryl acetate (1mg/mL in 100% EtOH, 12 mL/min) stream and a citrate buffer stream pH 7.5(10 mM sodium citrate, 1.6 μM citric acid, 36 mL/min) within the MIVMgeometry [FIG. 1(a)]. Dyes and CAI-1 were dissolved in the 100% EtOHsolution containing 1 mg/mL α-tocopheryl acetate at variousconcentrations ranging from 0.1-10% wt relative to the particle corethat comprised zein plus α-tocopheryl acetate. The resulting NPsuspension contained 20% EtOH (v/v) with a relative mass of zein tocasein to VitE of 6:3:1. To achieve different zein to casein ratios, theconcentration of casein in citrate buffer was adjusted while respectiveflow rates of zein and casein solutions were kept the same.

Nanoparticle Characterization

Using a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK), dynamiclight scattering (DLS) size measurements were performed on samplesdirectly after FNP. Samples were diluted with ultra-pure water to avoidmultiple scattering and analyzed at 25° C. using a detection angle of173°. The reported size is the intensity-weighted average diameter asreported by the Malvern deconvolution software in Normal Mode analysis.Samples for TEM were prepared by placing 5 μL of the nanoparticledispersion on an ultrathin carbon film on a holey carbon support film on400 mesh copper grid (Ted Pella, Inc., Redding, Calif.) and drying underambient conditions. The samples were imaged using a Philips CM100 TEM(Eindhoven, The Netherlands) operated at an accelerating voltage of 100kV.

Fluorescence of Nile Red-loaded Zein NPs

The optical properties of Zn NPs were characterized with a F-7000Fluorescence Spectrophotometer (Hitachi High Technologies America) aftertenfold NP dilution in miliQ water. Emission scans were measured betweena 530 nm to 800 nm window using a 500 nm excitation wavelength and 400mV PMT voltage. Excitation scans were measured between a 400 nm to 670nm window using a 690 nm emission wavelength at 400 mV PMT voltage.

Zein Nanoparticle Formulation

The encapsulation of poorly soluble actives into nanocarriers canenhance their bioavailability. Nanoparticle forming properties of zeinusing the FNP process (FIG. 1) were investigated. The herein presentedFNP process using GRAS materials results in quasi monodispersenanoparticles as evidenced by TEM [FIG. 1(b)]. The image depicts VitE-Accontaining zein/casein NPs (1.5:1 wt). Particles show a homogeneous sizedistribution of a darker core, comprising VitE and zein, and aresurrounded by a faint halo of sodium caseinate as stabilizer [FIG.1(b)].

The encapsulation of a lipid co-core resulted in an increased meanparticle diameter (from 127 nm to 199 nm) and PDI (from 0.008 to 0.026),while all other factors where held constant (FIG. 2a , formulations‘F-2’ and ‘F-3’). The addition of a basic amino acid, in this caselysine (‘F−’), decreased the particle mean diameter (84 nm) butincreased the PDI (0.076) despite the encapsulation of a lipid co-core.When an acidic moiety (malic acid) was incorporated in the formulation,the mean particle diameter (331 nm) increased. These results suggestthat the mean nanoparticle diameter and PDI can be tuned by modulatingthe amount of the lipid co-core that is encapsulated, the addition ofcharged moieties, and an adjustment in solution pH (FIG. 2), whilekeeping all other variables constant.

TPGS, Pluronic F68 and casein are FDA-approved surfactants. Their effecton particle characteristics and solution stability over time wereexamined by DLS. The size and PDI of zein NPs (ZNPs) was not affected bythe addition of Pluronic F68 or TPGS, relative to unstabilized zein inmilliQ water (FIG. 3a ). When zein encapsulated a hydrophobic corecomprising VitE-Ac (FIG. 3b ), however, the mean diameter and PDIincreased according to molecular weight of the surfactant relative tounstabilized ZNPs (ZNP<ZNP+TPGS<ZNP+F68). The use of casein assurfactant increased the PDI and reduced the mean ZNP diameter,regardless of whether VitE-Ac was encapsulated in the core or not. Theencapsulation of VitE-Ac at a relative mass to zein of 1:6 shows littleeffect on the particle size and PDI, while at a ratio of 5:6 by weight,the resulting particles have a larger mean diameter (FIG. 3c ). Theincrease of casein to zein ratio reduces mean particle size, whileincreasing the polydispersity (FIG. 3d ). These results demonstrate goodsize control and reproducibility of the NP formation process.

Impact of Surfactants on Formulation Stability

Premature nanoparticle dissolution or aggregation would lead to deliveryfailure based on either too fast or too slow release of the active. Zeincolloids aggregate in ionic solutions and during drying as result ofhydrophobic interactions unless stabilized by surfactants. Initially,nanoparticle formulations were screened for stability at physiologicconditions (PBS) and conditions relevant to the in vitro model of V.cholera. While TPGS and Pluronic F68 were capable of stabilizing ZNPs inmilliQ H₂O (PDI≤0.052), these particles began aggregating within 5minutes in PBS pH7.4 or LB50 (FIG. 15).

These formulations had completely aggregated within 5 hours. When ZNPswere stabilized by sodium caseinate, particles were stable in solutionover 5 hours and even showed reduced polydispersity when loaded with ≤1mg VitE-Ac (PDI_(t=5min)=0.109, PDI_(t=5hrs)=0.093). The stabilitybehavior of ZNPs was independent of the VitE-Ac core (Tables VI, VII).The stabilization of hydrophobic colloids can be achieved by stericstabilization, electrostatic repulsion, or both. Close to theisoelectric point of zein (˜pH6.8), steric stabilization with 2 wt. %TPGS or Pluronic F68 alone did not prevent aggregation. The isoelectricpoint of colloids containing sodium caseinate (˜pH4.6) and zein has beenreported to lie between the individual values and cause a surface chargereversal. The Zn/CAS stability at high ionic strength suggests aneffective coverage of the zein and the importance of the electrostaticeffect. Sodium caseinate sufficiently stabilized zein NPs manufacturedby FNP in 1×PBS pH7.4 and LB50 broth.Impact of Temperature on Nanoparticle Stability

Increased temperature, as is the case in the human body, may also leadto premature nanoparticle dissolution or aggregation and would thereforelead to delivery failure based on either too fast or too slow release ofthe active. Zein particles containing a lipid co-core, and stabilized byPLURONIC F68, were stable during a 2 hour heat ramp from 25° C. to 60°C., suspended in 0.01M HCl pH 2.0 (FIG. 4). The specific diameters andPDI did not change significantly between temperatures 25° C. (206 nm,0.045), 45° C. (210 nm, 0.005) and 60° C. (209 nm, 0.040).

Impact of Zn/CAS Ratio on Particle Characteristics and Stability

Various zein-to-casein ratios were produced to further investigateformulation stability in physiological environments relevant to oraldelivery. Zein to casein ratios 4:1, 2:1, and 1:1 FIG. 17 in PBS, pH2.0,LB50 and 2% bile salts were investigated. In phosphate buffer, particlesremained stable over 48 hrs with only minor changes in mean diameter orpolydispersity. In the small intestine, bile salts emulsify and degradelipids. While Zein/CAS/VitEAc NPs increased in size by about 40% whenexposed to 2% bile salts, particles retained colloidal stability over 48hrs FIG. 17. Specifically, Zein/CAS/VitEA NPs (2:1) showed bestcolloidal stability in PBS, 2% bile salts, LB50 and pH2.0 after 48 hrsFIG. 17.

Dye Encapsulation in Zein/CAS/VitEA Nanoparticles

The utility of an encapsulation method depends on its encapsulationcapacity and efficiency for various compounds. Successful encapsulationof dyes with different physicochemical properties (FIG. 5) has beendemonstrated (FIG. 6a ) for Nile Red (log P 3.65, Log D 4.46, pH7.4),Pyrene (log P 5.17, Log D 4.92 pH7.4) and Methyl Red (log P 4.91, Log D1.46 pH7.4). Due to its comparable hydrophobicity to CAI-1 (log P 4.35,log D 4.30 pH7.4), loading efficiency studies were conducted using NileRed as model compound. Nile Red was encapsulated at 0.2%-5 wt. %. At0.1% wt. Nile Red loading, the UV signal was below the detection limit,while at 10% wt. immediate aggregation of NPs was observed after FNP.Dye co-encapsulation with VitE-Ac at the core of zein/CAS NPs showed ared-shifted UV spectrum relative to free Nile Red solution in 100%,while the UV spectrum of free Nile Red in aqueous 20% EtOH showed astrong blue-shift (FIG. 7). This suggests the encapsulation of Nile Redinto Zein/CASNitEA NPs. Encapsulation efficiency of Nile Red inZn/CAS-VitE-Ac NPs was >98% for all stable formulations as determined byUV-Vis measurement of the flow through after centrifugal filtration(FIG. 7). With increasing Nile Red concentration in the core, the meannanoparticle diameter increases (FIG. 6b ). The florescence intensity isexpected to increase with higher dye loading per particle. This can beseen in FIGS. 6(c) and 6(d) for two different wavelengths (500 nmexcitation, 690 nm emission) and core loadings of 0.2-1% wt. However, atdye content higher than 1% wt., the fluorescence intensity decreases,which suggests a dye-quenching effect. Fluorescence quenching can derivefrom physical proximity of individual molecules, and in this case, itsuggests that Nile Red may preferentially accumulate in the hydrophobicVitE-Ac core.

Example 2—Composite Nanoparticles Stability and Redispersibility afterFreeze-Drying

1. Preparation

-   -   a. Buffer: 0.150 g Sodium citrate, and 1.5 mg citric acid in 50        mL milliQ H₂O, adjusted to pH7.5 with 1M NaOH, 1M HCl, then 0.2        μm filtered.    -   b. MIVM Line-in:        -   i. (1): 100% EtOH 2 mg/mL α-tocopherol (12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein+0.13 mg/mL F68 Pluronic            (12 mL/min)        -   iv. (4): Sodium citrate pH 7.5, 0.1M (36 mL/min)    -   c. Final NP concentration is 1.64 mg/mL in 20% EtOH.    -   d. NPs were either i) dialyzed against milliQ H₂O using a        cellulose dialysis tubing for 2 hours with one full buffer        exchange at 1 hr.    -   e. Particles were kept in milliQ H₂O 24 hrs before use at RT.    -   f. Freeze-drying excipient conditions:        -   i. 10 mM KH₂PO₄ pH7.5        -   ii. Sodium citrate buffer        -   iii. Maltodextrin 8% wt. in 0.1M sodium citrate buffer pH            7.5        -   iv. Maltodextrin 8% wt. in 10 mM KH₂PO₄ pH7.5        -   v. Sucrose 8% wt. in 10 mM KH₂PO₄ pH7.5        -   vi. Trehalose 8% wt. in 10 mM KH₂PO₄ pH7.5        -   vii. Pluronic F68 0.16% wt. 10 mM KH₂PO₄ pH7.5        -   viii. Maltodextrin 6% wt. in 0.1M sodium citrate buffer pH            7.5 and Pluronic F68 0.04% wt        -   ix. Maltodextrin 6% wt. in 10 mM KH₂PO₄ pH7.5 and Pluronic            F68 0.04% wt        -   x. Sucrose 6% wt. in 10 mM KH₂PO₄ pH7.5 and F68 0.04% wt        -   xi. Trehalose 6% wt. in 10 mM KH₂PO₄ pH7.5 and F68 0.04% wt    -   g. Samples were measured in liquid suspension (DLS) and frozen        on dry ice the same day (within 3 hours), then set to lyophilize        on tree freeze dryer (no precise temperature control)˜100 mTorr        for 72 hours. Samples were redispersed with milliQ water, to        original volume prior to freeze-drying, shortly vortexed, and        analysed by DLS.

2. Characterization of Particles

-   -   a. Dynamic light scattering: Used company recommended refractive        index settings for protein particles (RI: 1.450; Absorption:        0.010) in water (RI: 1.330; Viscosity: 0.8872 cP, 25° C.). Using        a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK), dynamic        light scattering (DLS) size measurements were performed on        samples directly after FNP and at a 10-fold dilution in milliQ        water (final EtOH content: <2% v/v). Samples were analyzed at        25° C. using a detection angle of 173°. The reported size is the        intensity-weighted average diameter as reported by the Malvern        deconvolution software in Normal Mode analysis.

3. Results

Phosphate buffer above 10 mM is known to destabilize and aggregate zeincolloidal particles. Zein was less stable in 10 mM KH₂PO₄ than in 0.1Msodium citrate. Additionally, freeze-concentration during lyophilisationlikely increased the molarity of the phosphate buffer, which wouldfurther drive aggregation of zein. However, the excipient formulation(10) comprising, 6% wt. sucrose, 0.04% wt. Pluronic F68 was able topreserve zein nanoparticles during freeze-drying and resuspension.Stability of the formulation would likely be further increased byincreasing sucrose content, and using sodium citrate buffer instead ofKH₂PO₄.

In the case of sodium caseinate as stabilizer, because of the higherzeta potential of the resulting nanocomposites, NPs are stable duringlyophilization and resuspension. Therefore, we demonstrate stabilityduring lyophilization for a formulation (using PLURONIC F68 assurfactant) that is more challenging to stabilize during lyophilizationand resuspension.

Example 3—Composite Nanoparticles Temperature Stability in AcidicConditions

1. Preparation

-   -   a. Buffer: 0.150 g Sodium citrate, and 1.5 mg citric acid in 50        mL milliQ H₂O, adjusted to pH7.5 with 1M NaOH, 1M HCl, then 0.2        μm filtered.    -   b. MIVM Line-in:        -   i. (1): 100% EtOH 2 mg/mL α-tocopherol (12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein+0.13 mg/mL F68 Pluronic            (12 mL/min)        -   iv. (4): Sodium citrate pH 7.5, 0.1M (36 mL/min)    -   c. Final NP concentration is 1.64 mg/mL in 20% EtOH.    -   d. NPs were dialyzed against milliQ H₂O using a cellulose        dialysis tubing for 2 hours with one full buffer exchange at 1        hr.    -   e. Particles were suspended in 0.1M HCl pH2.0 (simulating a        stomach pH environment) and particles were subjected to a        temperature ramp from 25° C. to 60° C., over a 2 hour period. NP        size and particle count rate were measured continuously by DLS.

2. Characterization of particles

-   -   a. Dynamic light scattering: Used company recommended refractive        index settings for protein particles (RI: 1.450; Absorption:        0.010) in water (RI: 1.330; Viscosity: 0.8872 cP, 25° C.). Using        a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK), dynamic        light scattering (DLS) size measurements were performed on        samples directly after FNP and at a 10-fold dilution in milliQ        water (final EtOH content: <2% v/v). Samples were analyzed at        25° C. using a detection angle of 173°. The reported size is the        intensity-weighted average diameter as reported by the Malvern        deconvolution software in Normal Mode analysis.

3. Results

The size of NPs were stable (diameter ˜209 nm+/−6 nm) during the 2 hrtemperature ramp from 25° C. to 60° C. in acidic conditions (0.01M HClpH 2.0). Moreover, PDI remained stable over time during heat ramp to 60°C. as illustrated in FIG. 4. Mean particle diameter and PDI attemperatures 25° C. (206 nm, 0.045), 45° C. (210 nm, 0.005), and 60° C.(209 nm, 0.040) did not vary significantly.

Example 4—Composite Nanoparticle Stability in PBS when Pretreated withDicarboxylic Acid

1. Preparation

-   -   a. Buffer: 0.150 g Sodium citrate, and 1.5 mg citric acid in 50        mL milliQ H₂O, adjusted to pH7.5 with 1M NaOH, 1M HCl, then 0.2        μm filtered.    -   b. MIVM Line-in:        -   i. (1): 100% EtOH 2 mg/mL α-tocopherol (12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein+0.13 mg/mL F68 Pluronic            (12 mL/min)        -   iv. (4): Sodium citrate pH 7.5, 0.1M (36 mL/min)    -   c. Final NP concentration is 1.64 mg/mL in 20% EtOH.    -   d. NPs were dialyzed against milliQ H₂O using a cellulose        dialysis tubing for 2 hours with one full buffer exchange at 1        hr.    -   e. Particles were suspended in 22% malic acid at pH13, and        cross-linked for 2 hours at room temperature (protocol adapted        from Reddy et al. 2009 AlChE “Alkai-catalyzed low temperature        wet crosslinking of plant proteins using carboxylic acids”).

2. Characterization of Particles

-   -   a. Dynamic light scattering: Used company recommended refractive        index settings for protein particles (RI: 1.450; Absorption:        0.010) in water (RI: 1.330; Viscosity: 0.8872 cP, 25° C.). Using        a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK), dynamic        light scattering (DLS) size measurements were performed on        samples directly after FNP and at a 10-fold dilution in milliQ        water (final EtOH content: <2% v/v). Samples were analyzed at        25° C. using a detection angle of 173°. The reported size is the        intensity-weighted average diameter as reported by the Malvern        deconvolution software in Normal Mode analysis.

3. Results

Malic acid treated NPs retained size better in 1×PBS relative tountreated NPs, which aggregated within 5 min of being suspended in1×PBS. Malic acid has previously shown to cross-link zein hydroxylgroups via intermediate cyclic anhydride on the acid catalyst thatcross-links with the polymer/peptide (Reddy N et al. Biotechnol Prog.2009). However, even malic acid treated particles aggregated over timein PBS.

Particle Type Size Dh (nm) PDI NPs in H₂O 174 0.023 Malic acid treated316.5 0.061 Malic acid treated in 1x PBS t = 0 282.3 0.203 Malic acidtreated in 1x 516.2 0.255 PBS t = 30

Example 5—Size Control of Zein Nanoparticles, Various Formulations

Various FNP-MIVM formulations achieve a different range of nanoparticlesize with low PDI.

1. Preparation

-   -   a. Buffer: 0.150 g Sodium citrate, and 1.5 mg citric acid in 50        mL milliQ H₂O, adjusted to pH7.5 with 1M NaOH, 1M HCl, then 0.2        μm filtered.    -   b. Formulation 1 (MIVM Line-in):        -   i. (1): 100% EtOH 1 mg/mL α-tocopherol (12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein+0.4 mg/mL Pluronic            F6+1.8 mg/mL D-Lysine (12 mL/min)        -   iv. (4): Sodium citrate pH 7.5, 0.1M (36 mL/min)    -   c. Formulation 2 (MIVM Line-in):        -   i. (1): 100% EtOH (12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein, 2 wt % Pluronic F68        -   iv. (4): Sodium citrate pH 7.5, 0.1M (36 mL/min)    -   d. Formulation 3 (MIVM Line-in):        -   i. (1): 100% EtOH 2 mg/mL α-tocopherol (12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein, 0.35 mg/mL Pluronic F68        -   iv. (4): Sodium citrate pH 7.5, 0.1M (36 mL/min)    -   e. Formulation 4 (MIVM Line-in):        -   i. (1): 100% EtOH 1 mg/mL α-tocopherol (12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein, 0.35 mg/mL Pluronic            F68, 1.6 mg/mL Malic Acid (12 mL/min)        -   iv. (4): Sodium citrate pH 7.5, 0.1M (36 mL/min)    -   f. NPs were either i) dialyzed agains milliQ H₂O using a        cellulose dialysis tubing for 2 hours with one full buffer        exchange at 1 hr, or ii) stored as is (in 20% EtOH) at either RT        or 4-8° C.

2. Characterization of particles

-   -   a. Dynamic light scattering: Used company recommended refractive        index settings for protein particles (RI: 1.450; Absorption:        0.010) in water (RI: 1.330; Viscosity: 0.8872 cP, 25° C.). Using        a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK), dynamic        light scattering (DLS) size measurements were performed on        samples directly after FNP and at a 10-fold dilution in milliQ        water (final EtOH content: <2% v/v). Samples were analyzed at        25° C. using a detection angle of 173°. The reported size is the        intensity-weighted average diameter as reported by the Malvern        deconvolution software in Normal Mode analysis.    -   b. pH was measured

F-1 F-2 F-3 F-4 Size (nm) 84 127 199 331 PDI 0.076 0.008 0.026 0.049

Results

As provided in the table above and FIG. 2, the FNP process using theMIVM mixer produced zein nanoparticle in with low polydispersity over awide range of sizes. DLS measurements recorded mean diameters rangingfrom 80 nm-330 nm, and individual particles ranging from 40 nm-700 nm.The PDI remained below 0.1 for the formulations tested. Lysine has a pKaof 10.5 and is a basic residue. Formulation 1 was slightly basic, whileFormulation 4 was slightly acidic. The charge of zein, as dependent onthe pH of the solution, therefore impacts the final size ofnanoparticles. The difference between formulation F-2 having no lipidcore and F-3 is the inclusion of a Vitamin-E core (at 2 mg/mL).

Example 6—Inverse Zein Nanoparticles Precipitated into Ethanol

Zein is an amphiphilic biopolymer that can be precipitated against anaqueous stream or an organic stream. Zein is insoluble in pure EtOH.Instead of precipitating into aqueous environment, where the hydrophobicresidues are predominantly on the particle interior, zein nanoparticlesare precipitated into organic (EtOH) to form a predominantly hydrophilicparticle interior with a hydrophobic exterior.

Preparation

-   -   a. MIVM Line-in:        -   i. (1): 100% EtOH (12 mL/min)        -   ii. (2): 100% EtOH (36 mL/min)        -   iii. (3): 60% EtOH of 11 mg/mL Zein (12 mL/min)        -   iv. (4): 100% EtOH (36 mL/min)    -   b. Final NP concentration is 1.375 mg/mL in 95% EtOH.    -   c. NPs were diluted 10-fold in 100% EtOH and measured using DLS.

Characterization of Particles

-   -   b. Dynamic light scattering: Used company recommended refractive        index settings for protein particles (RI: 1.450; Absorption:        0.010) in ethanol (RI: 1.360; Viscosity: 1.095 cP, 25° C.).        Using a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK),        dynamic light scattering (DLS) size measurements were performed        on samples directly after FNP and at a 10-fold dilution in 100%        EtOH (final aqueous content: <0.5% v/v). Samples were analyzed        at 25° C. using a detection angle of 173°. The reported size is        the intensity-weighted average diameter as reported by the        Malvern deconvolution software in Normal Mode analysis.

Results

Inverse zein nanoparticles were formed using the MIVM. The high supersaturation and flow rates allow for nucleation and growth of NPs at 95%EtOH concentration within the mixer. Without stabilizer, zein particleshad a mean diameter of 418 nm+/−36 nm, and a polydispersity of0.242+/−0.013 as illustrated in FIG. 8.

Example 7—Composite Nanoparticle Bioactivity

To minimize loss of precious actives, it was important to ensure minimalfree drug in solution after FNP. To identify the highest loadingefficiency for CAI-1 into Zn/CAS-VitE-Ac NPs, CAI-1 encapsulation wastested for 1×, 2×, 3×-fold mass of NPs at a constant amount of drug.Compositions and flow rates for the nanocarrier species in FIG. 9(a) areprovided in FIG. 19.

The encapsulation of CAI-1 did not impact the size or PDI of NPs.Changes in size and PDI could only be observed for the three-foldprotein per amount of drug formulation (FIG. 9a ). All formulationsshowed similarly high encapsulation as determined by in vitrobioactivity assay (data not shown). All parts of the formulation werenecessary for the formation of nanoparticles with defined size. NPs thatlacked either zein, or zein and casein did not form nanoparticles withcontrolled size (FIG. 9b ). FNP of CAI-1 alone yielded a polydisperseemulsion, which then agglomerated and settled out over time. There wasno apparent effect of CAI-1 or NPs on the growth of cells during thetime tested (FIG. 10a ).

The bioactivity of blank Zein/CAS/VitEA particles was not detectable(FIG. 10b ). The retentate of NPs containing CAI-1 showed highanti-bacterial activity, and was similarly efficacious as free drug inDMSO, and unfiltered NPs containing CAI-1 (FIG. 10b ). The flow-throughafter centrifugal filtration can be regarded as a correlate forencapsulation efficiency of CAI-1. Bioactivity of the flow through wasobserved only at very high concentrations, approximately1,000-10,000-fold higher than CAI-1 NPs. This suggests better than 98%encapsulation efficiency of CAI-1 during FNP. These data suggest thatCAI-1 loaded Zein/CAS/VitEA NPs prepared by FNP, are a feasible andefficacious low-cost GRAS therapeutic with potential as efficaciouscholera prophylactic.

Example 8—Zein Co-Precipitation with Biomolecules, including NucleicAcids and Proteins

DNA-Zein Nanocarrier Preparation

-   -   a. Buffer: 0.150 g Sodium citrate, and 1.5 mg citric acid in 50        mL milliQ H₂O, adjusted to pH7.5 with 1M NaOH, 1M HCl, then 0.2        μm filtered.    -   b. MIVM Line-in:        -   i. (1): 62.5 m/mL Salmon Sperm DNA in 0.1M sodium citrate            buffer pH7.5 (12 mL/min)        -   ii. (2): dH₂O (36 mL/min)        -   iii. (3): 60% EtOH of 12 mg/mL Zein (12 mL/min)        -   iv. (4): dH₂O (36 mL/min)    -   Particles were collected after one-step Flash Nanoprecipitation        using the MIVM geometry.

BSA-Zein Nanocarrier Preparation

-   -   c. Buffer: 0.150 g Sodium citrate, and 1.5 mg citric acid in 50        mL milliQ H₂O, adjusted to pH7.5 with 1M NaOH, 1M HCl, then 0.2        μm filtered.    -   d. MIVM Line-in:        -   i. (1): 0.1 mg/mL BSA-FITC in 0.1M sodium citrate buffer            pH7.5 (12 mL/min)        -   ii. (2): dH₂O (36 mL/min)        -   iii. (3): 60% EtOH of 12 mg/mL Zein (12 mL/min)        -   iv. (4): dH₂O (36 mL/min)

Characterization of Particles

-   -   a. Dynamic light scattering: Used company recommended refractive        index settings for protein particles (RI: 1.450; Absorption:        0.010) in water (RI: 1.330; Viscosity: 0.8872 cP, 25° C.). Using        a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK), dynamic        light scattering (DLS) size measurements were performed on        samples directly after FNP and at a 10-fold dilution in milliQ        water (final EtOH content: <2% v/v). Samples were analyzed at        25° C. using a detection angle of 173°. The reported size is the        intensity-weighted average diameter as reported by the Malvern        deconvolution software in Normal Mode analysis.    -   b. Zeta potential: Using a Zetasizer Nano-ZS (Malvern        instruments, Malvern, U.K.), dynamic light scattering (DLS) size        measurements were performed on samples after FNP and dialysis.        For zeta potential measurements, samples were diluted with 20 mM        sodium chloride to a 10 mM salt concentration.

Results

-   -   Zein combines with salmon sperm DNA during FNP to form 152 nm        particles with low polydispersity (PDI=0.062). Moreover, the        formed nanocarriers incorporated 4.7 μg of DNA per 1 mg of zein        at solution pH of 7.5, zein feed concentration of 12 mg/ml and        relative mass ratio zein:DNA of 200:1. FIG. 10 provides        additional parameters of DNA encapsulation by zein. FIG. 11        illustrates DNA encapsulation and loading efficiency into zein        containing colloids according to some embodiments described        herein.    -   Zein combines with BSA-FITC during FNP to form 194 nm particles        with low PDI=0.073. Additionally, the formed nanocarriers        incorporated 7.5 μg of BSA-FITC per 1 mg of zein at solution pH        of 7.5, zein feed concentration of 12 mg/ml and relative mass        ratio zein:BSA-FITC of 16:1. FIG. 12 illustrates BSA-FTIC        encapsulation and loading efficiency into zein containing        colloids according to some embodiments described herein.        FIG. 13 illustrates the effect of additives or stabilizers,        having an isoelectric point below solution pH, on the size and        PDI of resulting colloids according to some embodiments        described herein. FIG. 14 illustrates the effect of additives or        stabilizers, having an isoelectric point below solution pH, on        the zeta potential of the resultant nanocarrier colloids        according to some embodiments described herein.

Example 9—Zein Shell, No Core Nanoparticles, Will Encapsulate NegativelyCharged Biomolecules, Such as DNA or RNA

-   -   Rationale: The isoelectric point of alpha-zein occurs at pH 6.8,        and the isoelectric point of DNA occurs at approximately pH 5.0.        At pH 5.5, zein is positively charged and DNA (and RNA) is        negatively charged. Based on opposite net charge, the        encapsulation of DNA (and RNA) into zein will be enhanced. The        polar side chains on zein will interact with DNA. This can be        true also at other pH ranges that are higher or lower than        pH6.0. The resulting particles, will be charge stabilized, in        that DNA (and RNA) will act in a similar fashion as sodium        caseinate, that when combined with zein, alters the overall-zeta        potential of the construct (nanoparticle), which will afford        colloidal stability at physiologic conditions based on        electrostatics. These formulations may include other        surfactants. Ratios of zein:DNA and zein:RNA (and versions        thereof, such as but not limited to: pDNA (plasmids), mRNA,        siRNA, etc.) of 100:1 to 250:1 are expected to provide best        particle formation propensity.

1. Preparation

-   -   a. Buffer 1: 0.150 g Sodium citrate, and 1.5 mg citric acid in        50 mL milliQ H₂O, adjusted to pH 5.5 (but may range from pH 3.0        to pH 7.5) with 1M NaOH, 1M HCl, then 0.2 μm filtered.    -   b. Buffer 2: 10 mM Tris, 1 mM EDTA, pH 6.0 (but may range from        pH 5.5 to pH 7.5)    -   c. MIVM Line-in:        -   i. (1): 100% EtOH (12 mL/min; but may range from 2 ml/min to            12 mL/min)        -   ii. (2): Sodium citrate pH 5.5, 0.1M (36 mL/min; but may            range from 2 mL/min to 36 mL/min)        -   iii. (3): 70% EtOH, pH 5.5 of 12 mg/mL Zein (12 mL/min; but            may range from 2 ml/min to 12 mL/min)        -   iv. (4): 15-40 ug DNA/mL (best 25 ug DNA/mL) Tris-EDTA pH            6.0, 0.01M (36 mL/min; but may range from 2 mL/min to 36            mL/min)    -   d. The resultant solution is adjusted to pH10, using 1M NaOH, 1M        HCl, and is then dialyzed against milliQ dH₂O pH10 for 2 hours        with 1 complete buffer change.

2. Characterization of particles

-   -   a. Dynamic light scattering: Will use company recommended        refractive index settings for protein particles (RI: 1.450;        Absorption: 0.010) in water (RI: 1.330; Viscosity: 0.8872 cP,        25° C.). Using a Zetasizer® Nano-ZS (Malvern instruments,        Malvern, UK), dynamic light scattering (DLS) size measurements        will be performed on samples directly after FNP and at a 10-fold        dilution in milliQ water (final EtOH content: <2% v/v). Samples        will be analyzed at 25° C. using a detection angle of 173°. The        reported size will be the intensity-weighted average diameter as        reported by the Malvern deconvolution software in Normal Mode        analysis.

3. Results

-   -   a. The expected zeta potential of zein encapsulated DNA (and        RNA) at pH 10 is approximately −70 mV. This will impart        colloidal stability based on charge.    -   b. Particles with zein:DNA ratios of 100:1 to 250:1 are expected        to be colloidally stable in 1×PBS (pH7.4) for 3 hours or longer.    -   c. Encapsulation efficiencies are expected to average about 40%        (milligram DNA per gram of zein) but may be higher than 60%, and        lower than 30%.

Example 10—Zein Shell, No Co-Core Nanoparticles, Encapsulating Proteins(>40 amino acids) and Peptides (<40 amino acids) by Side-GroupInteractions

-   -   Rationale: Zein can be used to encapsulate proteins (>40 amino        acids) and peptides (<40 amino acids) using FNP. One possible        application is the protection of enzymes from degrading        conditions in the GI tract. For example, catalase (protein; pI        pH 5.4; Samejima et al. 1962) or superoxide dismutase (protein;        pI pH 4.95; Bannister et al. 1971) have been encapsulated using        conventional zein coacervation method by magnetic stirring        (Sugmun Lee et al Int. J. Pharm 2013). The synthetic peptide        Desmopressin used as hematologic agent (log P −5.82, log        D_(pH7.4) −7.34; amides, amines, hydroxyl) has been encapsulated        in zein. Here, the driving force for encapsulation is not net        hydrophobicity or net charge of the molecule, but interactions        of protein or peptide side groups with the side groups of zein.        We can encapsulate proteins using the herein presented FNP        method.

1. Preparation

-   -   a. Buffer 1: 0.150 g Sodium citrate, and 1.5 mg citric acid in        50 mL milliQ H₂O, adjusted to pH 7.5 (but may range from pH 6.0        to pH 8.0) with 1M NaOH, 1M HCl, then 0.2 μm filtered.    -   b. MIVM Line-in:        -   i. (1): 100% EtOH (12 mL/min; but may range from 2 ml/min to            12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min; but may            range from 2 mL/min to 36 mL/min)        -   iii. (3): 60% EtOH of 12 mg/mL Zein (may range from 5 mg/mL            to 15 mg/mL)+0.15 mg/mL F68 Pluronic (or TPGS; might range            from 0.10 mg/mL to 3 mg/mL) at a flow rate of 12 mL/min (but            may range from 2 mL/min to 12 mL/min)        -   iv. (4): 10-40 μg protein/mL in sodium citrate pH 7.5, 0.1M            (36 mL/min; but may range from 2 mL/min to 36 mL/min).    -   c. NPs will be dialyzed against milliQ H₂O using a cellulose        dialysis tubing for 2 hours with one full buffer exchange at 1        hr.

2. Characterization of particles

-   -   a. Dynamic light scattering: Will use company recommended        refractive index settings for protein particles (RI: 1.450;        Absorption: 0.010) in water (RI: 1.330; Viscosity: 0.8872 cP,        25° C.). Using a Zetasizer® Nano-ZS (Malvern instruments,        Malvern, UK), dynamic light scattering (DLS) size measurements        will be performed on samples directly after FNP and at a 10-fold        dilution in milliQ water (final EtOH content: <2% v/v). Samples        will be analyzed at 25° C. using a detection angle of 173°. The        reported size will be the intensity-weighted average diameter as        reported by the Malvern deconvolution software in Normal Mode        analysis.

3. Results

-   -   a. Particles are expected to have a mean diameter of 180 nm, but        may range from 90 nm to 400 nm.    -   b. Particles are expected to be stable in low ionic aqueous        buffers, an approximate pH range from pH 2.0 to pH 9.0, and        milliQ H₂O.    -   c. Encapsulation efficiencies are expected to average about 40%        (milligram protein per gram of zein) but may be higher than 60%,        and lower than 30%.

Example 11—Zein Shell, No Co-Core Nanoparticles, Will Encapsulate SmallMolecules with Charged Groups

-   -   Rationale: Zein can be used to encapsulate small molecules with        charged groups, and with or without a net dipole moment using        FNP. For example, the anti-inflammatory drug Mesalazine (MW        153.1 g/mol; log P 0.46, log D_(pH7.4) −1.98; primary amine,        hydroxyl, carboxylic acid) has been encapsulated into        microparticles using conventional zein coacervation method        (Esther Lau et al. Pharmaceutics 2013). Here, the driving force        for encapsulation is not net hydrophobicity or net charge of the        molecule, but interactions of charged functional groups on the        small molecule with the side groups of zein. We can encapsulate        proteins using the herein presented FNP method.

1. Preparation

-   -   a. Buffer 1: 0.150 g Sodium citrate, and 1.5 mg citric acid in        50 mL milliQ H₂O, is adjusted to pH 7.5 (but may range from pH        6.0 to pH 8.0) with 1M NaOH, 1M HCl, then 0.2 μm filtered.    -   b. MIVM Line-in:        -   i. (1): 100% EtOH (12 mL/min; but may range from 2 ml/min to            12 mL/min)        -   ii. (2): Sodium citrate pH 7.5, 0.1M (36 mL/min; but may            range from 2 mL/min to 36 mL/min)        -   iii. (3): 60% EtOH of 12 mg/mL Zein (may range from 5 mg/mL            to 15 mg/mL)+0.15 mg/mL F68 Pluronic (or TPGS; might range            from 0.10 mg/mL to 3 mg/mL) at a flow rate of 12 mL/min (but            may range from 2 mL/min to 12 mL/min)        -   iv. (4): 1 mg molecule/mL (but may range from 0.01 mg/mL to            2 mg/mL) in sodium citrate pH 7.5, 0.1M (36 mL/min; but may            range from 2 mL/min to 36 mL/min)

2. Characterization of Particles

-   -   a. Dynamic light scattering: Will use company recommended        refractive index settings for protein particles (RI: 1.450;        Absorption: 0.010) in water (RI: 1.330; Viscosity: 0.8872 cP,        25° C.). Using a Zetasizer® Nano-ZS (Malvern instruments,        Malvern, UK), dynamic light scattering (DLS) size measurements        will be performed on samples directly after FNP and at a 10-fold        dilution in milliQ water (final EtOH content: <2% v/v). Samples        will be analyzed at 25° C. using a detection angle of 173°. The        reported size will be the intensity-weighted average diameter as        reported by the Malvern deconvolution software in Normal Mode        analysis.

3. Results

-   -   a. Particles are expected to have a mean diameter of 120 nm, but        may range from 80 nm to 400 nm.    -   b. Particles are expected to be stable in low ionic aqueous        buffers, an approximate pH range from pH 2.0 to pH 9.0, and        milliQ H₂O.    -   c. If sodium caseinate is used as surfactant, particles will be        stable in physiologic buffer conditions for at least 3 hours.    -   d. Encapsulation efficiencies are expected to average about 10%        (milligram molecule per gram of zein) but may be higher than        30%, and lower than 5% wt/wt.

Various embodiments of the invention have been described in fulfillmentof the various objectives of the invention. It should be recognized thatthese embodiments are merely illustrative of the principles of thepresent invention. Numerous modifications and adaptations thereof willbe readily apparent to those skilled in the art without departing fromthe spirit and scope of the invention.

The invention claimed is:
 1. A method of preparing a compositenanoparticle composition comprising: providing a zein solution stream;providing an organic solvent fluid stream comprising at least oneadditive; and providing at least one buffer fluid stream, wherein thezein solution stream, organic solvent fluid stream and buffer fluidstream are separately delivered to a chamber for mixing at one or morerates sufficient to flash precipitate composite nanoparticles includingthe additive encapsulated by a shell comprising the zein, wherein theadditive is selected from the group consisting of a pharmaceuticalcomposition, nutraceutical composition, food composition, beveragecomposition, agro active composition, taste-masking composition,biomolecular composition, personal care composition and cosmeticcomposition, and wherein the composite nanoparticles exhibit apolydispersity less than 0.3 in PBS buffer for a time period of at least5 hours.
 2. The method of claim 1, wherein the composite nanoparticleshave an average size of 10 nm to 500 nm.
 3. The method of claim 2,wherein the composite nanoparticles exhibit a polydispersity of lessthan 0.15.
 4. The method of claim 1, wherein the organic solvent fluidstream comprises a plurality of additives.
 5. The method of claim 4,wherein the plurality of additives are encapsulated by the shellcomprising zein.
 6. The method of claim 1, wherein the zein solutionstream, organic solvent fluid stream and buffer fluid stream aresimultaneously mixed in the chamber.
 7. The method of claim 1, whereinzein solution stream, organic solvent fluid stream and buffer fluidstream are delivered to the chamber by independent feed lines.
 8. Themethod of claim 1, wherein the one or more rates is at least 10 mL/min.9. The method of claim 8, wherein the zein solution stream and theorganic solvent fluid stream are delivered at a rate of 10-20 mL/min,and the buffer fluid stream is delivered at a rate of 30-40 mL/min. 10.The method of claim 1, wherein the zein solution stream comprises zeinsolubilized in a hydroalcoholic solution.
 11. The method of claim 10,wherein the zein solution stream further comprises one or morestabilizers.
 12. The method of claim 11, wherein the stabilizerscomprise one or more surfactants.
 13. The method of claim 11, whereinthe one or more stabilizers are incorporated into or onto the compositenanoparticles.
 14. The method of claim 13, wherein the one or morestabilizers are encapsulated by or adsorbed onto the shell comprisingthe zein.
 15. The method of claim 13, wherein the one or morestabilizers are incorporated in or adsorbed onto the shell comprisingthe zein.
 16. The method of claim 13, wherein the one or morestabilizers are encapsulated by the shell and incorporated in the shellcomprising the zein.
 17. The method of claim 1, wherein the buffer fluidstream comprises one or more stabilizers.
 18. The method of claim 17,wherein the one or more stabilizers comprise casein.
 19. The method ofclaim 1, wherein the additive is present in the composite nanoparticlesin an amount of 0.1-60 weight percent.
 20. The method of claim 1,further comprising extracting organic solvent from the nanoparticlecomposition for reuse in preparation of additional compositenanoparticles.
 21. The method of claim 1 having an additiveencapsulation efficiency of 40-100 percent.
 22. The method of claim 21,wherein the additive encapsulation efficiency is 70 to 95 percent. 23.The method of claim 1, wherein the organic solvent is selected from thegroup consisting of ethanol, tetrahydrofuran, dimethylsulfoxide, anddimethylformamide.
 24. A method of preparing a composite nanoparticlecomposition comprising: providing a zein solution stream; providing anorganic solvent fluid stream comprising at least one additive; andproviding at least one buffer fluid stream, wherein the zein solutionstream, organic solvent fluid stream and buffer fluid stream areseparately delivered to a chamber for mixing at one or more ratessufficient to flash precipitate composite nanoparticles including theadditive encapsulated by a shell comprising the zein, whereinencapsulation efficiency of the additive is 90 percent to 100 percent.25. A method of preparing a composite nanoparticle compositioncomprising: providing a zein solution stream; providing an organicsolvent fluid stream comprising at least one additive; and providing atleast one buffer fluid stream, wherein the zein solution stream, organicsolvent fluid stream and buffer fluid stream are separately delivered toa chamber for mixing at one or more rates sufficient to flashprecipitate composite nanoparticles including the additive encapsulatedby a shell comprising the zein, wherein the additive is present in thecomposite nanoparticles in an amount of 1 to 20 weight percent.
 26. Amethod of preparing a composite nanoparticle composition comprising:providing a zein solution stream; providing an organic solvent fluidstream comprising at least one hydrophobic additive; and providing atleast one buffer fluid stream, wherein the zein solution stream, organicsolvent fluid stream and buffer fluid stream are separately delivered toa chamber for mixing at one or more rates sufficient to flashprecipitate composite nanoparticles including the hydrophobic additiveencapsulated by a shell comprising the zein.